The proposed research will study the biochemical pathogenesis of Leigh's disease (LD) or subacute necrotizing encephalomyelopathy. Brains of patients with LD have a specific deficiency of thiamine triphosphate, with a relative accumulation of thiamine diphosphate. Other reported etiologies include deficiencies of either pyruvate carboxylase (PC) or pyruvate dehydrogenase (PDH) activities. In the first year of the research we were able to demonstrate a normal PC activity in patients with LD. The activity of PDH complex was studied in fibroblasts from several LD patients and found to be normal. We have concluded that deficiencies of PDH or PC are probably not valid etiologies for LD as previously suggested. The next step is to continue the purification of the inhibitor associated with LD. Observations to date suggest that it can be purified using standard column chromotography techniques. We will continue to seek patients with LD and collect large amounts of urine from these patients for study. In addition, we will be studying the normal metabolism of thiamine in the central nervous system. One year ago we discovered an acid active thiamine diphosphatase in beef brain. This enzyme will be purified and characterized.